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Published in final edited form as:
Environ Microbiol Rep. 2010 August ; 2(4): 534–540. doi:10.1111/j.1758-2229.2009.00098.x.
Variation in Pseudonocardia antibiotic defence helps govern
parasite-induced morbidity in Acromyrmex leaf-cutting ants
Michael Poulsen1,*, Matías J. Cafaro1,2, Daniel P. Erhardt1, Ainslie E. F. Little1, Nicole M.
Gerardo3, Brad Tebbets4, Bruce S. Klein4, and Cameron R. Currie1
1Departments of Bacteriology, University of Wisconsin, Madison, Microbial Sciences Building,
1550 Linden Dr., Madison, WI 53706, USA
2Department
of Biology, University of Puerto Rico – Mayaguez, PO Box 9012, Mayaguez, Puerto
Rico 00681
4Medical
Microbiology and Immunology, University of Wisconsin, Madison, Microbial Sciences
Building, 1550 Linden Dr., Madison, WI 53706, USA
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3Department
of Biology, Emory University, O. Wayne Rollins Research Center, 1510 Clifton Road
NE, Room 2006, Mail Stop #1940-001-1AC, Atlanta, GA 30322, USA.
Summary
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Host–parasite associations are potentially shaped by evolutionary reciprocal selection dynamics, in
which parasites evolve to overcome host defences and hosts are selected to counteract these
through the evolution of new defences. This is expected to result in variation in parasite-defence
interactions, and the evolution of resistant parasites causing increased virulence. Fungus-growing
ants maintain antibiotic-producing Pseudonocardia (Actinobacteria) that aid in protection against
specialized parasites of the ants’ fungal gardens, and current evidence indicates that both
symbionts have been associated with the ants for millions of years. Here we examine the extent of
variation in the defensive capabilities of the ant–actinobacterial association against Escovopsis
(parasite-defence interactions), and evaluate how variation impacts colonies of fungus-growing
ants. We focus on five species of Acromyrmex leaf-cutting ants, crossing 12 strains of
Pseudonocardia with 12 strains of Escovopsis in a Petri plate bioassay experiment, and
subsequently conduct subcolony infection experiments using resistant and non-resistant parasite
strains. Diversity in parasite-defence interactions, including pairings where the parasites are
resistant, suggests that chemical variation in the antibiotics produced by different actinobacterial
strains are responsible for the observed variation in parasite susceptibility. By evaluating the role
this variation plays during infection, we show that infection of ant subcolonies with resistant
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd
*
For correspondence. [email protected]; Tel. (+1) 608 890 0237; Fax (+1) 608 262 9865. .
Supporting information Additional Supporting Information may be found in the online version of this article:
Fig. S1. Cultivar–Escovopsis Petri plate bioassay. The average number of days the 12 Acromyrmex-associated Escovopsis isolates
took to overgrow cultivars and sporulate when presented with cultivars from the same 12 colonies in an in vitro Petri plate bioassay.
Different colours indicate the average number of days: white: 14+ days, light yellow: 12–13 days, yellow: 10–11 days, orange: 8–9
days, and red: 6–7 days (n = 3).
Fig. S2. Relationships between Pseudonocardia size and Escovopsis degree of inhibition. The association between Pseudonocardia
size and the zone of inhibition (ZOI) in the Pseudonocardia–Escovopsis pairings (r = 0.12; R2 = 0.0152).
Table S1. Results of the statistical evaluation of Escovopsis– cultivar bioassay. The top section of the table gives the nested model,
having cultivar and Escovopsis clones nested within their respective ant species origins. The bottom section of the table gives the
analyses excluding ant origins, and evaluating only the symbiont clones individually and their interaction. Both analyses were
performed under a gamma distribution and significance values evaluated with likelihood ratio statistics for a Type III analysis.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the corresponding author for the article.
Poulsen et al.
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parasite strains results in significantly higher parasite-induced morbidity with respect to garden
biomass loss. Our findings thus further establish the role of Pseudonocardia-derived antibiotics in
helping defend the ants’ fungus garden from the parasite Escovopsis, and provide evidence that
small molecules can play important roles as antibiotics in a natural system.
Introduction
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The biology of all living organisms, including humans and human-domesticated plants and
animals, is influenced by parasites (Jaenike, 1978; Price et al., 1986; Ewald, 1994; Cohen,
2000; Palumbi, 2001). The constant selective pressures imposed by parasites on hosts have
made them a major force influencing evolution and species diversification (Jaenike, 1978;
Ewald, 1994; Cohen, 2000; Palumbi, 2001). On an evolutionary time scale, natural host–
parasite associations are shaped, at least in part, by arms race-like dynamics: parasites
evolve to overcome host defences and hosts are selected to counteract these through the
evolution of new defences (Van Valen, 1973; Hamilton, 1980). In humans, the dynamics of
host– parasite interactions are modified through the application of bacteria-derived (Clardy
et al., 2005) antimicrobial chemicals. However, the efficacy of these compounds in human
medicine and agriculture is continuously threatened by the emergence of resistant parasite
phenotypes, which arise as a consequence of selective pressures imposed by the applied
antimicrobials (Cohen, 2000; Palumbi, 2001; Davies, 2007). Recent work has revealed that
some insects also employ bacteria-derived antibiotics for protection against parasites
(fungus-growing ants: Currie et al., 1999a; Oh et al., 2009; European beewolves: Kaltenpoth
et al., 2005; and Southern Pine beetles: Scott et al., 2008). Theory predicts that the target
parasites of the bacteria-derived antibiotic compounds should evolve resistance, but the
presence and impact of such resistant parasites have not been evaluated in any of these
natural systems. Here, we determine the potential for the evolution of parasite resistance by
determining whether: (i) there is variation in parasite resistance, and (ii) this variation leads
to differential parasite success. Fungus-growing ants in the tribe Attini (Hymenoptera:
Formicidae) engage in an obligate mutualism with fungi they cultivate for food (Agaricales:
Lepiotaceae and Pterulaceae) (Chapela et al., 1994; Munkacsi et al., 2004; Schultz and
Brady, 2008). This beneficial ant–fungus symbiosis is parasitized by microfungi in the
genus Escovopsis that attack and consume the ants’ fungal garden (Hypocreales:
Ascomycota) (Currie et al., 1999a; 2003b; Reynolds and Currie, 2004). The cultivated fungi
can suppress the growth of Escovopsis strains not known to infect them in nature, but cannot
inhibit isolates of naturally occurring parasites (Gerardo et al., 2006a,b; Gerardo and
Caldera, 2007; Supporting information; Fig. S1). Instead, to defend their fungus gardens
from Escovopsis, the ants rely on behavioural removal of Escovopsis spores and mycelium
(Currie and Stuart, 2001), secretions from worker’s metapleural glands (Bot et al., 2002;
Fernández-Marín et al., 2009), and mutualistic antibiotic-producing filamentous bacteria in
the genus Pseudonocardia (Actinomycetales: Pseudonocardiaceae; Currie et al., 1999b;
Cafaro and Currie, 2005) (Fig. 1A). The relative contribution of these different defence
mechanisms likely depends on the genus of fungus-growing ant. In the ant genus
Acromyrmex, artificially removing Pseudonocardia from the ant cuticle results in
significantly larger negative effects of Escovopsis, indicating that Pseudonocardia is an
important defence against these parasites (Currie et al., 2003a). Actinobacteria in other
genera have also been isolated from ant fungus gardens (Mueller et al., 2008; Haeder et al.,
2009), and in culture some strains have been shown to produce small molecules that inhibit
the growth of Escovopsis (Haeder et al., 2009). However, it remains to be established if
these bacteria are more than transient community members (i.e. being isolated because they
are present as non-metabolizing inocula introduced to the garden with the leaf material
added by workers), or instead are capable of growth and production of these small molecules
within the garden matrix (see Results and discussion). Individual Acromyrmex ant colonies
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appear to associate with a single Pseudonocardia strain (Poulsen et al., 2005), which is
persistently present within nests (at least in laboratory colonies) for extended periods of time
(years) (••. Marsh, M. Poulsen and C.R. Currie, unpubl. data), but there is genetic diversity
in this bacterial symbiont both within and between ant species (Cafaro and Currie, 2005;
Poulsen et al., 2005; Mikheyev et al., 2008). The between-colony diversity in ant-associated
Pseudonocardia leads to the prediction that different Pseudonocardia strains have different
inhibitory capabilities against different Escovopsis strains (Currie et al., 2006).
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In order to build on our knowledge of the role of Pseudonocardia in the association with
fungus-growing ants, we examine how differences in inhibitory capabilities between
different Pseudonocardia strains affect Escovopsis-induced morbidity. We do this by linking
observed Pseudonocardia–Escovopsis interactions in vitro to in vivo defensive capabilities
during subcolony infection. Capitalizing on the ability to culture the microbial symbionts
associated with Acromyrmex leaf-cutting ants, we examine interactions between the
antibiotic-producing bacteria and the target parasites in a bioassay experiment, crossing
strains associated with 12 colonies from five species of Acromyrmex leaf-cutting ants (Fig.
1A and B). Based on the variation in Escovopsis–Pseudonocardia (parasite-defence)
interactions in vitro, we test the prediction that the inhibitory capabilities of Pseudonocardiaderived antibiotics correlate with parasite-induced morbidity in fungus-growing ant nests in
vivo. We do this by performing an infection experiment pairing colonies with Escovopsis
strains susceptible or resistant to the antibiotics produced by the resident bacterium.
Results and discussion
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Our symbiont pairings, evaluating the variation in parasite-defence interactions in a Petri
plate bioassay experiment of Pseudonocardia and Escovopsis from 12 colonies distributed
across five Acromyrmex species, revealed abundant variation in suppression of Escovopsis
(Fig. 1B). The degree of Escovopsis inhibition observed depended on the specific
combination of strains of Pseudonocardia and Escovopsis paired. The reason for this
variation is likely a combination of (i) the susceptibility of Escovopsis, (ii) compound
differences between Pseudonocardia strains, (iii) compound production rate differences
between Pseudonocardia strains, and (iv) potential differences between bacterial strains in
their composition of compounds when more than a single compound is produced. This is
supported by our findings of a statistically strong effect of the interaction between
Pseudonocardia inhibitory capabilities and the susceptibility of Escovopsis (likelihood ratio
statistics for a Type III two-way ANOVA: χ2 = 202.5, d.f. = 104, P < 0.0001) (Table 1).
The analysis further identified significant strain effects of both Escovopsis (likelihood ratio
statistics for a Type III nested ANOVA: χ2 = 14.50, d.f. = 7, P = 0.0429) and
Pseudonocardia (χ2 = 114.9, d.f. = 7, P < 0.0001) (Table 1). The variation observed
included pairings in which the parasite Escovopsis exhibited significant resistance to the
Pseudonocardia-derived antibiotics. Escovopsis growth was completely suppressed by the
antibiotic-producing Actinobacteria in 54.2% of bioassay pairings, but Escovopsis was not
suppressed, indicating antibiotic resistance, in 11.8% of the bioassay pairings (Fig. 1B).
Despite abundant variation in interactions within and between ant species, there were
notable effects of the ant species origin of both symbionts (Pseudonocardia χ2 = 41.41, d.f.
= 4, P < 0.0001; Escovopsis: χ2 = 21.21, d.f. = 4, P = 0.0003; Table 1). Our analysis also
indicated a possible dose effect (χ2 = 9.59, d.f. = 1, P = 0.002), but this correlation
explained only 1.5% of the variation (r = 0.12; Fig. S2). The presence of resistance in the
parasite and variation in bacteria-derived chemistry suggests that interactions on this finer
phylogenetic scale shape host–parasite dynamics within this system. In fact, 45.3% (29 out
of 64) of pairings between symbionts isolated from the two sympatric Acromyrmex species
(A. echinatior and A. octospinosus) showed weak or no inhibition [zone of inhibition (ZOI)
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< 1.0 cm] in the bioassay, indicating that resistant Escovopsis phenotypes do infect leafcutting ant nests in nature.
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In order to examine whether the patterns of inhibition of Escovopsis observed reflect general
antifungal properties of the Pseudonocardia strains tested, we performed a supporting
experiment testing the antifungal effects of the 12 strains against a strain of Candida
albicans (strain 1002, see Supporting information for more details). We used the same
experimental Petri plate bioassay set-up as in the Pseudonocardia–Escovopsis pairings,
except that C. albicans was inoculated in a liquid suspension, and found that none of the 12
Pseudonocardia strains displayed visible inhibition of C. albicans. Although we cannot rule
out that inhibition of C. albicans could occur at higher concentrations of Pseudonocardiaderived secondary metabolites, this set-up allows for a direct comparison with the
Pseudonocardia–Escovopsis assay. This suggests that the variation in Escovopsis inhibition
between Pseudonocardia strains is not a result of differences in the levels of general
antifungal activity between different bacterial strains (for details, see Supporting
information).
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Based on the Petri plate bioassay results, we predicted that the extent of parasite-induced
garden biomass loss would depend upon the combination of Pseudonocardia and Escovopsis
parasite strain. To test this prediction, we performed a subcolony experiment pairing six
different ant colonies with three different Escovopsis strains, choosing pairings that included
the range of antagonistic displays of Pseudonocardia symbionts towards Escovopsis in vitro
(Fig. 1B). Escovopsis impact was evaluated as fungus garden mass loss 1 week after
Escovopsis application. Escovopsis-induced garden biomass loss indeed depended on the
combination of the resident Pseudonocardia associated with the host ant colony and the
strain of Escovopsis (Fig. 1C). The in vivo evaluation revealed that Escovopsis rapidly
overgrew all cultivar fragments in the absence of ants (one-way ANOVA: F7,752 = 11 240, P
< 0.0001; Table 2). In the presence of ants, the impact of Escovopsis was strongly correlated
with the inhibition, or lack thereof, displayed in the in vitro bioassay by the resident
Pseudonocardia bacterium, indicating that reactions in vitro represent the effectiveness of
bacterial defence in vivo (F2,123 = 9.66, P < 0.0001) (Fig. 1C; Table 2). Of particular interest
were situations where Escovopsis strains were only weakly inhibited by the bacterium in
vitro (ZOI < 1.0 cm; 25.7% of the pairings), as these combinations, on average, showed
virulence levels in vivo that were comparable to pairings with strongly resistant Escovopsis
strains (Fig. 1C) (F1,83 = 0.2819, P = 0.5969). A two-way ANOVA confirmed the isolated
effects of ant colony (F5.135 = 5.74, P < 0.0001) and Escovopsis treatment (F6,135 = 23.53, P
< 0.0001), and further showed a marginally non-significant interaction between the two
(F15.135 = 1.64, P = 0.071) (Table 2). The results of this experiment indicate that the
inhibitory capabilities of the antimicrobials produced by the resident Pseudonocardia
bacterium directly influence the impact of Escovopsis during colony infections. Thus, strains
of the garden parasite exhibiting in vitro resistance to Pseudonocardia secondary metabolites
cause greater morbidity to the ants’ fungus garden in vivo, confirming that the symbiont
bioassay pairings reflect interactions within colonies.
Further, our infection experiment indicates that even strains of Escovopsis exhibiting some,
but not strong, resistance in vitro (i.e. growth suppression < 1.0 cm ZOI) cause higher levels
of fungus garden morbidity in vivo. This indicates that more than a third of the parasitedefence pairings screened in the bioassay exhibit a degree of resistance to the antibiotics
produced by the bacteria that would result in increased garden morbidity. Importantly,
resistance was present in Escovopsis– Pseudonocardia combinations found in nature, and
even occurred, although only rarely, in pairings between symbionts from the same
individual ant colony (Fig. 1B). While it may appear counterintuitive to find surviving
colonies inhabited by resistant parasite strains, other defences in the ants (e.g. grooming and
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weeding; Currie and Stuart, 2001) likely compensate for this lack of efficient
Actinobacteria-defence and thus help prevent complete colony collapse in such rare cases.
Despite the presence of resistant parasite strains, a large portion of pairings in the bioassay
experiments exhibited strong suppression of the parasite (62.5%) (Fig. 1B), and infection
with parasite strains susceptible to the Actinobacteria exhibited less garden biomass loss
(Fig. 1C). This implies that the costs incurred by the ants to maintain Pseudonocardia,
through specialized exocrine glands that secrete the substrate for Pseudonocardia growth
(Poulsen et al., 2003; Currie et al., 2006), are outweighed by the benefits obtained by the
ability of Pseudonocardia to suppress Escovopsis during colony infections.
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Naturally occurring resistant parasite strains, like those identified in this study, should
impose a selective pressure on the ant host for the evolution of novel defences. The ant–
Pseudonocardia association appears to maintain efficient defence against Escovopsis despite
the evolution of antibiotic resistance. The mechanisms behind the successful application of
Actinobacteria-derived antibiotics by fungus-growing ants are unclear, but several scenarios
can be envisioned. The first conceivable scenario is that parasite-defence dynamics in the
fungus-growing ant symbiosis are shaped by antagonistic co-evolution, involving the
evolution of resistance in the parasite, counteracted by the evolution of novel compounds by
Pseudonocardia. Specifically, this posits that the selective pressure imposed by Escovopsis
on Pseudonocardia results in the Pseudonocardia-derived antibiotics evolving in parallel
with the target parasites. In turn, the antibiotic secretions by Pseudonocardia targeted at
Escovopsis strains are expected to impose a selective pressure on Escovopsis to evolve
resistance (Cohen, 2000; Palumbi, 2001; Davies, 2007). The presence of such antagonistic
co-evolution would result in variability in the chemistry of Pseudonocardia secretions, as our
bioassay pairings suggest (Fig. 1B). The relatively short generation time of bacteria may
allow for a fast rate of change, but our understanding of changes in the metabolic capacities
of the bacterium residing in individual nests is, as of yet, limited. Such change either may be
restricted to include only minor modifications of existing secondary metabolites, and not
major and elaborate alterations, or may involve the possibility of acquiring new antibioticcoding gene clusters through horizontal gene transfers (Ginolhac et al., 2005; Jenke-Kodama
et al., 2005; Schmitt and Lumbsch, 2009). An alternative mechanism for rapid modifications
is the acquisition of a novel Pseudonocardia strains, with novel secondary metabolites, by an
ant colony. Co-phylogenetic patterns between Pseudonocardia and fungus-growing ants
suggest relatively frequent switching of the Pseudonocardia symbiont between species, and
even genera (Poulsen et al., 2005; Mikheyev et al., 2008), as well as several acquisitions of
Pseudonocardia from free-living (non-fungus-growing ant-associated) counterparts over the
evolutionary history of the association (Cafaro and Currie, 2005). Both evolution of novel
Pseudonocardia antibiotics and acquisition of novel antibiotic-producing bacteria from the
environment likely play a role in mediating host–pathogen dynamics in this system.
It is generally recognized that just because one microbe inhibits another in Petri plate
challenges, this may imply, but does not prove, that the compound produced causes
inhibition in nature (Waksman, 1961; Davies, 2006; Clardy et al., 2009). In fact, it has been
argued that what we consider antibiotics do not function as such in nature, but instead are
signalling compounds (Davies, 2006; Yim et al., 2007). Recently, it has been shown that
Actinobacteria genera other than Pseudonocardia (mostly Streptomyces) have the potential
to inhibit Escovopsis (Mueller et al., 2008; Haeder et al., 2009). However, these studies are
only supported by bioassays, not infection experiments, and thus implies, but does not show,
that these Actinobacteria could play a similar role in the fungus-growing ant symbiosis. In
contrast, the role of Pseudonocardia-derived antibiotics for dealing with Escovopsis
infections is now supported through both removal and infection experiments (Currie et al.,
2003a) and through the evaluation of the role inhibitory capabilities play in governing
parasite-induced morbidity (this study). For the two main species of Acromyrmex focused
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on in this study, A. octospinosus and A. echinatior, it is well established that Pseudonocardia
is vertically transmitted between generation by incipient queens (Currie et al., 1999a), and
that specific Pseudonocardia strains are associated with these ant species (Cafaro and Currie,
2005; Poulsen et al., 2005). In addition, the same Pseudonocardia strain can be isolated from
the exoskeleton of workers in individual Acromyrmex colonies maintained in the lab for
several years, indicating consistent and persistent associations with Pseudonocardia (••.
Marsh, M. Poulsen and C.R. Currie, unpubl. data). Haeder and colleagues (2009) also
isolated Pseudonocardia as the main bacterium from workers of A. octospinosus and A.
echinatior; they only found Streptomyces in the fungus garden matrix. Although these
Streptomyces strains potentially inhibit Escovopsis in Petri plates, it remains to be
determined if they represent more than transient microbes that do not actively metabolize
within the ant symbiosis (see Caldera et al., 2009).
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Given the metabolic capacity of Actinobacteria to produce antibiotic (e.g. Demain, 1999), in
addition to the relatively short generation time of bacteria allowing for faster rates of
change, it is perhaps not surprising that the defensive mutualist in the association is an
Actinobacterium. This suggests that the ant–Pseudonocardia mutualism is an example of
symbiosis as evolutionary innovation: the ants benefit from the mutualism by gaining access
to a source of rapidly changing metabolites with potent antimicrobial properties. The
development of novel antibiotics by Pseudonocardia strains may evolve in response to
Escovopsis parasites, thereby counteracting the evolution of parasite resistance. The
availability of potent antibiotic producers to modify their secondary metabolism and/or the
ants’ ability to acquire new sources of antibiotics from other ant-associated or free-living
bacteria may have allowed for a more efficient defence against Escovopsis than would have
been achieved by the ants alone. Long-term antibiotic use in ants may only have remained
stable and efficient over time due to their ability to access microbes from the environment
that harbour diverse antimicrobial properties. Whether or not novel chemistries are obtained
from the resident ant-associated bacterium, or via acquisition forming novel associations,
our findings illustrate the benefits of this type of defensive mutualism in the ability to
counteract resistance evolution in the target parasite.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
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We thank M.R. Bergsbaken, P. Foley and D.J. Molinaro for laboratory assistance, T.R. Schultz for assistance with
species identifications of some of the ants used in the study, R. Chappell, J.A. Dawson, B.R. Larget and T.-L. Lin
for suggestions on statistics, CALS Statistical Consulting for providing facilities to perform analyses on the
bioassays, the Smithsonian Tropical Research Institute (STRI) for providing facilities to work in Panama, the
Autoridad Nacional del Ambiente y el Mar (ANAM) for sampling and export permissions, and S. Adams, E.
Caldera, A. Pinto-Tomas, G. Suen, J.M. Thomas and two anonymous reviewers for constructive comments on the
manuscript. This work was supported by Lundbeckfonden to M.P. and NSF (CAREER-747002) to C.R.C.
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Fig. 1.
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A. Leaf-cutting ant worker with Pseudonocardia-covered cuticle. Micrograph of an
Acromyrmex octospinosus major worker illustrating the abundance of mutualistic bacteria
on the ant cuticle. B. Acromyrmex-associated Pseudonocardia–Escovopsis bioassay. Results
of a symbiont pairing bioassays between 12 strains of Pseudonocardia (vertical) and
Escovopsis (horizontal) associated with five species of Acromyrmex leaf-cutting ants. These
included three Argentinean species: A. niger (colony code CC030327-02), A. hispidus fallax
(SP030327-01 and UGM030327-02) and A. laticeps (UGM030330-04), and two
Panamanian species: A. echinatior (Ae291, Ae292, Ae295 and CC031212-01) and A.
octospinosus (CC011010-04, CC031210-22, ST040116-01, UGM020518-05). Pure culture
Pseudonocardia were point inoculated in the centre of plates containing PDA without
antibiotics and were grown for 3 weeks until reaching a diameter of c. 1–1.5 cm, after which
Escovopsis was inoculated at the edge of the plate. Plates were examined bi-weekly and
when a clear zone of inhibition (ZOI) had formed in a given pairing (typically within 2–3
weeks after Escovopsis inoculation), the ZOI between symbionts (Cafaro and Currie, 2005)
was recorded. Each box represents the average ZOI (n = 3) of a given pairing and different
colours indicate the degree of inhibition: white: ZOI = 0 cm, light yellow: ZOI = 0.01–0.50
cm, yellow: ZOI = 0.51–1.0 cm, orange: ZOI = 1.01–1.5 cm, and red: ZOI > 1.51 cm.
Bioassay pairings enclosed in boxes represent the pairings from which subcolonies were
created and inoculated with Escovopsis (Fig. 1C). C. Subcolony evaluation of Escovopsis
virulence. The in vivo virulence of Escovopsis measured as fungus garden biomass loss
when miniature colonies, created using a standard set-up (Bot et al., 2001; Poulsen and
Boomsma, 2005) with 80 mg of fungus garden and two major Acromyrmex workers
covered with the Pseudonocardia bacterium (abundance scores 9–12 in Poulsen et al., 2003),
were exposed to Escovopsis infection. Subcolonies were housed in 60 ml plastic cups with
small holes in the lids to provide air, moist tissue in the bottom to assure high humidity, and
a folded oak leaf to provide forage for the ants. To assure that Escovopsis spore inoculations
did not differ between subcolonies, we quantified spore concentrations in aqueous
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suspensions containing filter-sterilized water and 0.05 μl Tween 20 per ml water prior to
application, and subsequently inoculated individual subcolonies with an amount of
suspension corresponding to 11 500 ± 252 spores (mean ± SE; n = 17). Negative control
subcolonies were applied the same volume of filter-sterilized water and Tween 20 without
Escovopsis spores. Fungus garden weight was measured 1 week after Escovopsis
application. Fungus garden loss is averaged across three strength levels of in vitro
Escovopsis inhibition by Pseudonocardia (strong: ZOI > 1.0 cm, n = 48; weak: ZOI = 0.01–
1.0 cm, n = 42; or no: ZOI = 0 cm, n = 36; Fig. 1B).
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Table 1
Results of the statistical evaluation of the within-Acromyrmex Pseudonocardia–Escovopsis bioassay.
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χ2
d.f.
P
Nested model
Pseudonocardia strain (ant species)
114.9
7
< 0.0001
Escovopsis strain (ant species)
14.50
7
0.0429
Ant species of Pseudonocardia
41.41
4
< 0.0001
Ant species of Escovopsis
21.21
4
0.0003
Ant species of Pseudonocardia * ant
species of Pseudonocardia
28.14
15
0.0207
Size of Pseudonocardia
9.590
1
0.002
Pseudonocardia strain
233.8
11
< 0.0001
Escovopsis strain
77.83
11
< 0.0001
Pseudonocardia strain * Escovopsis
strain
202.5
104
< 0.0001
Size of Pseudonocardia
1.930
1
Two-way ANOVA
0.1616
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Due to the non-normality of data and the high sensitivity of imputing, zero-values in log-linear models, we employed a generalized linear model in
which zero-values were replaced with the value, 0.00001 and significance of terms in the models was determined through likelihood ratio statistics
for Type III ANOVAs. The analysis was performed in SAS v. 9.1.3 (SAS Institute, 1994). Two models were built: the first model contained the
respective ant species from which Pseudonocardia and Escovopsis strains originated from, Pseudonocardia and Escovopsis strains nested within
their ant species origins, the interaction term between the ant species symbionts originated from, and the size of Pseudonocardia at the time
inhibition of Escovopsis was scored (top section). Since this nested design did not allow for an evaluation of the interaction between
Pseudonocardia and Escovopsis strains, we performed a separate analysis including only Pseudonocardia and Escovopsis strains and their
interaction (bottom section).
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Table 2
Statistical analyses of subcolony evaluation of Escovopsis virulence in Acromyrmex.
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d.f.
F
P
Two-way ANOVA
Colony origin
5,135
5.740
< 0.0001
Escovopsis strain
6,135
23.53
< 0.0001
15,135
1.640
0.071
11,240
7.752
< 0.0001
2,123
9.660
< 0.0001
1,83
0.2819
0.5969
Escovopsis strain
5,120
2.532
0.0323
Ant colony
5,120
6.331
< 0.0001
Colony origin * Escovopsis strain
One-way ANOVAs
Absence of ants
No, weak or strong actinobacterial
inhibition in vitro
No or weak actinobacterial inhibition
in vitro
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The top section of the table shows the results of a two-way ANOVA evaluating the overall effects of Escovopsis on subcolonies of Acromyrmex
ants in vivo. The bottom section represents four one-way ANOVAs testing the isolated effects of (i) ant presence in subcolonies, (ii) whether the
Pseudonocardia symbiont associated with the ant colony exhibited strong (ZOI > 1.0 cm), weak (ZOI = 0–0.9 cm) or no (ZOI = 0 cm) in vitro
inhibition of the Escovopsis strain, (iii) whether there was a difference in Escovopsis virulence between subcolonies harbouring Pseudonocardia
strains exhibiting weak (0 < ZOI < 0.9 cm) or no (ZOI = 0) in vitro inhibition of the Escovopsis strain, (iv) individual Escovopsis strains, and (v)
ant colony.
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